1. Field of the Invention
The present invention relates to cell biology. In particular, this invention is directed to methods used to detect metabolic activity of biological samples based on their ability to consume oxygen.
2. Description of Relevant Art
Early knowledge about the metabolism of a new chemical entity (NCE) helps in the drug development process by providing important information for the selection of a lead compound from among a number of substances pharmacologically equally effective in their therapeutic response. In vitro metabolic studies may give information regarding the metabolic stability, toxicity, potential drug-drug interactions and the identification of the enzymes necessary for the metabolism of a compound.
The results of in vitro metabolic studies of an NCE determine to a great extent its future as a drug candidate. For example, if an NCE is rapidly metabolized, its bioavailability in vivo is most probably too low for it to be a drug. In vitro metabolic studies may reveal that an NCE can inhibit the binding of other drugs to enzymes, possibly resulting in prolonged pharmacological effects or an increased risk of drug toxicity. These results may be reason to eliminate it as a drug candidate.
NCEs and other chemical entities are mainly metabolized by enzymes in the liver, kidneys, gastrointestinal tract, skin, and lungs. Chemical entity-metabolizing enzymes are found in the endoplasmic reticulum of cells in these tissues and are classified as microsomal enzymes. There are 2 types of drug-metabolizing enzymes: phase I enzymes, or mixed function oxidases, which catalyze predominantly oxidation, reduction, and hydrolysis; and phase II enzymes, which catalyze glucuronidation, sulfation, or acetylation.
The majority of phase I metabolism is catalyzed by the cytochrome P-450 enzymes, which are heme-containing, membrane-bound proteins. These enzymes, found at highest concentration in the hepatocytes, biotransform lipophilic chemical entities to more polar compounds that can be excreted by the kidneys. The cytochrome P450's catalyze an oxidative reaction that is characterized by the oxidation of a substrate using atmospheric oxygen (O2).
Several known in vitro assays utilize oxidation reactions to measure oxygen consumption in order to assess, for example, if a particular enzyme is capable of metabolizing a test compound. One known method involves exposing a chemical entity to one or more preparations of enzymes and then placing the samples in chemical communication with a luminescent compound such as ruthenium dye which is quenched in the presence of oxygen. As oxidation of the chemical entity occurs, oxygen in the sample will be consumed leading to an increase in fluorescence. The increase in fluorescence is taken as an indication of metabolization.
The fluorescent intensity data, ordinarily read by a fluorometer, may be analyzed by a variety of methods to ascertain whether a particular chemical entity is being metabolized. For instance, as further oxygen is consumed over time, the amount of fluorescence correspondingly increases. Accordingly, after a predetermined period of time has elapsed (e.g. 1 hour), the magnitude of fluorescence emission from a sample having the metabolized chemical entity may be much greater than the magnitude of fluorescence emission from a control, such as a sample not containing the chemical entity. The value of the last reading taken for the sample can be compared with the control. If the sample value is above the control value at the final time point, the sample is identified as a positive sample. However, if the last value taken of the sample is below the control value, the sample is identified as a negative sample.
Although this “endpoint” detection” method can generally be effective in identifying positive and negative samples, it's not uncommon for this method to incorrectly identify a positive sample as being negative. Discrimination between metabolizable or nonmetabolizable chemical entities involves detecting small differences between fluorescence signals. The sample value may not be significantly larger than the control value after replicate variances are taken into account at any particular time point even though the sample may be, in fact, positive.
Also, it may be desirable to use only a small amount of a chemical entity which may make it difficult to generate an oxygen consumption rate which is sufficiently higher than the control value even though the chemical entity may be effectively metabolized by particular enzymes. At any given time point, there may be no apparent variation between a control value and the sample, resulting in the conclusion that metabolism is not occurring.
Other methods have been developed as well. In one method, the overall change in magnitudes of a sample reading is calculated and compared to a known value having a magnitude indicative of a positive result. Accordingly, if the magnitude of change is greater than the predetermined value, the sample is identified as a positive sample. On the other hand, if the magnitude of change is less than the predetermined value, the sample is identified as a negative sample.
Although this method may be more effective than the endpoint detection method discussed above, certain flaws in this method also exist. For example, if a sample contains a highly metabolizable chemical entity, the amount of oxygen consumption may reach a maximum at the time the initial reading is taken, and increase very little throughout the duration of the reading period. In this event, the change which occurs between the initial readings and final readings is minimal even though the sample is positive. Hence, the sample may be incorrectly identified as a negative sample.
Accordingly, a continuing need exists for a method to analyze data representative of readings using luminescence based systems to accurately discriminate between small differences in luminesence signals.